War-related injuries, fractures and surgeries on the limbs can all lead to the development of complex regional pain syndrome (CRPS), a potentially chronic and profoundly debilitating state. Currently there is no consensus on the pathogenesis of this enigmatic syndrome, and the treatment options for CRPS are very limited. The identification of the underlying disease mechanisms and preclinical evaluation of potential approaches to therapy are essential in order for us to treat or even prevent this disorder from occurring. Recent observations suggest that CRPS may be an autoimmune disorder. Evidence includes the identification of autoantibodies, reports of response to intravenous immunoglobulin (IVIG) treatment, genetic associations with HLA loci and the overabundance of Langerhan's antigen presenting cells in the skin of CRPS patients. Our own studies indicate that CRPS-like changes in the well-validated mouse fracture/cast model are reliant upon antibody production, and rely on intact neuropeptide signaling. The central hypothesis guiding our work is that that limb trauma causes the neurally-regulated production of autoantibodies and the regional expression of autoantigens leading to immune complex formation, complement fixation and, ultimately, pain sensitization. The primary objective of this proposal is to define the autoimmune contribution to CRPS and identify potential treatments targeting this disease mechanism. The specific aims are; 1) To delineate the contribution of autoimmunity to CRPS and understand the regulation of autoantibody production. Our multi-faceted experimental approach includes the use of longitudinal immunoglobulin isotype analysis, B-cell deficient mice, chemical and genetic neuropeptide depletion, chemical sympathectomy and serum transfer experiments. Human sample analysis will be pursued as well. 2) To identify the role of autoimmune activation of the complement cascade in supporting pain-related changes and neurite loss in the CRPS model. In these experiments we will use pharmacological agents and transgenic mice to identify the roles of C5a in supporting pain in the CRPS model. We will go on to measure the production of the C5b-9 membrane attack complex, and its role in damaging sensory neurites. The regulation of complement activation by peptidergic and sympathetic signaling will be defined, and 3) To identify the autoantigens involved in CRPS-related autoimmunity using murine and human CRPS patient samples. We hypothesize that the targets of autoimmunity include proteins differentially up-regulated in CRPS limbs, and that neuropeptide and sympathetic signaling are required for autoantigen expression and autoantibody production. We will focus on the expression of 5 proteins tentatively identified using liquid chromatography/mass spectrometry and the time course of autoantibody production against these targets. These experiments will establish a rigorous foundation for further exploring the mechanisms of post- traumatic autoimmunity, greatly improve our understanding of CRPS, identify specific molecular targets for future CRPS trials, and potentially suggest novel mechanisms supporting other enigmatic chronic pain syndromes. This proposal has the potential to identify novel pharmacologic treatments that could reduce pain and enhance the quality of life of CRPS patients.